The first step in biofilm formation is bacterial attachment to a surface. This attachment is mediated by components on the cell surface as well as the surface itself. Understanding the chemical interactions involved in attachment is an important part of preventing biofilm-related illness. Several proteins have been implicated in bacterial attachment and biofilm initiation, but their behavior on surfaces is poorly understood. Moreover, few experimental techniques exist that are able to characterize surface-bound protein behavior. This project investigates the properties of two biofilm-related proteins in Streptococcus epidermidis, Aap and AtlE, as they interact with surfaces. Recently developed approaches using NMR spectroscopy will be employed to study the structure and orientation of these proteins on various nanoparticle surfaces. Nanoparticles offer a significant increase in surface to volume ratio compared to macroscopic surfaces, and recent evidence suggests that nanoparticle curvature does not substantially alter the nature of protein-surface interactions. This makes nanoparticles an attractive system for studying protein-surface interactions. In this work, nanoparticles are used to model surfaces of materials made of glass, plastic, and titanium (commonly used in medical devices), as well as surfaces exposed to host extracellular matrix proteins. Three specific aims are proposed: (1) To identify the structural mode of interaction between biofilm-related proteins and surfaces, NMR-based hydrogen deuterium exchange, chemical labeling, and relaxation measurements will be used to characterize Aap and AtlE domains bound to surfaces. (2) To understand how biofilm protein competition influences surface binding, mixtures of Aap and AtlE domains will be studied, monitoring simultaneous surface binding in real time. (3) To determine how biofilm proteins bind to chemically passivated surfaces, we will explore the protein properties (e.g. charge, size) of Aap and AtlE domains that modulate binding to surfaces coated with PEG and Tween-20. Treatment with PEG is a common strategy for reducing protein binding, but this does not prevent binding entirely, and the reason why is not clear. Biofilms represent a major cause of hospital-associated infection in the US, and this project will lead to a better understanding of the early stages of bacterial attachment. This project applies novel and innovative techniques to study the chemical basis of adsorption of Aap and AtlE, and the results will be directly relevant to other bacterial biofilms as well. The mechanistic details revealed by this project will be useful in understanding how biofilms form, and such insights could ultimately lead to better approaches for inhibiting the formation of biofilms on surfaces.